Variability of laccase activity in the white-rot basidiomycete Pleurotus ostreatus

The production of laccase in liquid cultures of the white-rot fungusPleurotus ostreatus was highly variable. During the first days of cultivation, the relative variability was as high as 80–100% and it decreased to 30% in the course of cultivation. The main source of variability was assumed to be the independent development of enzyme activity in individual cultures. Cultures with high laccase production showed also high production of the other ligninolytic enzyme—Mn-dependent peroxidase. The variability was probably due to the source of inoculum, deactivation of the enzyme in culture liquid and genetic variations among the cultures. Variability of laccase activities was lower during solid-state fermentation on wheat straw and during the growth in nonsterile soil.     

Kinetic differences of purified laccases from six Pleurotus ostreatus strains

AIMS: Enzyme kinetics of purified laccases from six different Pleurotus ostreatus strains were determined in the oxidation of syringaldazine, guaiacol and ABTS. METHODS AND RESULTS: Significant differences in the kinetic constants were found. Catalytic activity (kcat) ranged from 19 to 941 U mg(-1) for syringaldazine, from 18 to 1565 U mg(-1) for ABTS, and from 4 to 44 U mg(-1) for guaiacol. The apparent affinity constants (KM) also showed significant differences between the different strains, from 12 to 52 micromol l(-1) for syringaldazine, from 8 to 79 micromol l(-1) for ABTS, and from 0.46 to 6.61 mmol l(-1) for guaiacol. No differences were found either on the effect of increasing concentrations of organic solvent (acetonitrile) or on the activity pH profile. The temperature profile was the same for all the P. ostreatus strains, except for the IE8 strain, which seems to be more sensitive to temperature. The kinetic and stability data from the six P. ostreatus strains were also compared with those obtained from other white rot fungi, Coriolopsis gallica and Trametes versicolor, showing clear differences. CONCLUSION: The different P. ostreatus isolates showed different kinetic constants. SIGNIFICANCE AND IMPACT OF THE STUDY: The different enzymatic properties of laccases from various P. ostreatus strains should be considered for a potential industrial or environmental application.     

Growth and laccase production by Pleurotus ostreatus in submerged and solid-state fermentation

Pleurotus ostreatus showed atypical laccase production in submerged vs. solid-state fermentation. Cultures grown in submerged fermentation produced laccase at 13,000 U l⁻¹, with a biomass production of 5.6 g l⁻¹ and four laccase isoforms. However, cultures grown in solid-state fermentation had a much lower laccase activity of 2,430 U l⁻¹, biomass production of 4.5 g l⁻¹, and three laccase isoforms. These results show that P. ostreatus performs much better in submerged fermentation than in solid-state fermentation. This is the first report that shows such atypical behavior in the production of extracellular laccases by fungi.     

Optimization of some variables that affect the synthesis of laccase by Pleurotus ostreatus

The influence of initial pH, concentration of yeast extract, inducer, type of enzyme releaser and buffer system on the composition of a medium for laccase production by Pleurotus ostreatus DM-1513 was investigated. A 2⁵ full factorial experimental design was initially employed to evaluate the effects of these variables on the enzyme synthesis. Data analysis showed that low pH and high yeast extract concentration values, as well as the absence of both an inducer and a buffer system, had positive effects on the secreted enzyme levels, whereas the type of enzyme releaser did not have a significant effect. The highest levels of laccase activity (489–540?U/l) were obtained in optimization experiments using media with initial pH between 6.0 and 6.5 and yeast extract concentrations of 0–0.25%     

Recombinant expression of Pleurotus ostreatus laccases in Kluyveromyces lactis and Saccharomyces cerevisiae

Heterologous expression of Pleurotus ostreatus POXC and POXA1b laccases in two yeasts, Kluyveromyces lactis and Saccharomyces cerevisiae, was performed. Both transformed hosts secreted recombinant active laccases, although K. lactis was much more effective than S. cerevisiae. rPOXA1b transformants always had higher secreted activity than rPOXC transformants did. The lower tendency of K. lactis with respect to S. cerevisiae to hyperglycosylate recombinant proteins was confirmed. Recombinant laccases from K. lactis were purified and characterised. Specific activities of native and recombinant POXA1b are similar. On the other hand, rPOXC specific activity is much lower than that of the native protein, perhaps due to incomplete or incorrect folding. Both recombinant laccase signal peptides were correctly cleaved, with rPOXA1b protein having two C-terminal amino acids removed. The availability of the established recombinant expression system provides better understanding of laccase structure–function relationships and allows the development of new oxidative catalysts through molecular evolution techniques.     

Comparison of three fungal laccases from Rigidoporus lignosus and Pleurotus ostreatus: correlation between conformation changes and catalytic activity

The conformation changes in solution of three fungal laccases in different environmental conditions were examined by circular dichroism (CD) and electron paramagnetic resonance (EPR) spectroscopy. CD measurements indicate that the secondary structure of proteins depends slightly on the pH or ionic strength, though the presence of salt could interfere in the molecular recognition process between substrates and enzymes. The enzymes, however, are highly destabilized by prolonged exposure to low pH or high temperature. The observed unfolding of the proteins coincides with their inactivation and, in some cases, with precipitation. On the other hand, these conditions do not determine the disruption of the geometric arrangement of their metal centres, and this fact suggests that these centres represent the more stable core of the proteins.     

Yellow laccase from the fungus Pleurotus ostreatus D1: Purification and characterization

Yellow laccase was isolated from a solid-phase culture of the fungus Pleurotus ostreatus D1 and characterized. It is a copper-containing enzyme with a molecular weight of 64 kDa. Its lacks an absorption spectrum maximum at 610 nm, a result which is characteristic of fungal laccases and corresponds to the presence of type I copper atoms. The optimum pH values for the enzyme are determined. They prove to be 7.0 for syringaldazine, 8.0 for pyrocatechol, and 4.0 for 2,2′-azine-bis-(3-ethylbenzothiazoline-6-sulfonate and 2,6-dimethoxyphenol. Kinetic parameters (K _m and V _max) for oxidation of these substrates are determined. The effect of inhibitors (SDS, 2-mercaptoethanol, and EDTA) on the activity of the enzyme is studied. It is shown that yellow laccase from Pleurotus ostreatus D1 in the absence of a mediator oxidizes anthracene to anthraquinone to 95%.     

Yellow laccase from the fungus Pleurotus ostreatus D1: purification and characterization

Yellow laccase was isolated from a solid-phase culture of the fungus Pleurotus ostreatus D1 and characterized. It is a copper-containing enzyme with molecular weight 64 kDa. Its absorption spectrum lacks the maximum at 610 nm, characteristic of fungal laccases and corresponding to type I copper atom. The optimum pH values for the enzyme were determined. They proved to be: 7.0 for syringaldazine, 8.0 for pyrocatechol, and 4.0 for 2,2'-azine-bis-(3-ethylbenzothiazoline-6-sulfonate and 2,6-dimethoxyphenol. Kinetic parameters (Km and Vmax) for oxidation of these substrates were determined. The effect of inhibitors (SDS, 2-mercaptoethanol, and EDTA) on the activity of the enzyme was studied. It was shown that yellow laccase from Pleurotus ostreatus D1 oxidized anthracene to anthraquinone by 95% without any mediator.     

启动子替代构建糙皮侧耳漆酶高产菌株

POXC是糙皮侧耳合成最多的一种漆酶。应用启动子替代技术,用构巢曲霉的甘油醛-3-磷酸脱氢酶基因（gpd）启动子替代POXC基因的启动子,构建了超量表达POXC糙皮侧耳转化子。转化子中POXC基因表达量比出发菌株提高了0.72–3倍。在PDA平板培养、PD摇瓶培养和棉籽壳试管培养条件下,转化子漆酶活力显著提高,比出发菌株提高了1.5倍以上。用棉籽壳栽培,转化子菇产量比出发菌株提高了16.2%,培养料中木质素含量比出发菌株减少21%。结果表明,应用高效启动子替代能够显著提高糙皮侧耳漆酶基因的表达量、漆酶活力及其木质纤维素降解能力。     

Manganese peroxidase isoenzymes produced by Pleurotus ostreatus grown on wood sawdust

The white rot basidiomycete Pleurotus ostreatus produces two manganese peroxidase (MnP) isoenzymes when grown in solid stationary conditions on poplar sawdust, whereas a lower production of these same enzymes is observed on fir sawdust. Addition of Mn(2+) to poplar culture resulted in a threefold increase of MnP activity; the same addition to fir culture was able to increase tenfold the MnP production. The two MnP isoenzymes (MnP2 and MnP3) were purified from P. ostreatus poplar culture. The isoenzymes differ in their pI values, molecular masses, and N-terminal sequences. MnP3 has the same N-terminal sequence as that of a P. ostreatus MnP previously reported. Both isoenzymes exhibit Mn(2+)-dependent and Mn(2+)-independent peroxidase activities when tested on phenolic substrates. The gene coding for the new isoenzyme MnP2 was cloned and sequenced and the promoter region analyzed. Furthermore, the chromosomal localization of all known P. ostreatus genes was determined.     

Molecular determinants of peculiar properties of a Pleurotus ostreatus laccase: Analysis by site-directed mutagenesis

A comparison of laccase sequences highlighted the presence of a C-terminal extension of sixteen amino acids in POXA1b laccase – that represents the most thermostable isoenzyme among Pleurotus ostreatus laccases and exhibits a notable stability at alkaline pH (t_1/2 at pH 10 = 30 days) – whereas this tail is missing in the other analysed laccases from basidiomycetes. Site-directed mutagenesis experiments allowed us to demonstrate a role of the C-terminal tail of POXA1b in affecting its catalytic and stability properties. The truncated mutants lose the high stability at pH 10, while they show an increased stability at pH 5. The effect of substituting the residue Asp205 of POXA1b with an arginine was also analysed in the mutant POXA1bD205R. Following the mutation POXA1bD205R, a remarkable worsening of catalytic properties along with a decrease of substrate affinity and of enzyme stability were found. It was demonstrated that introducing Arg205 mutation in a highly conserved region perturbs the structural local environment in POXA1b, leading to a large rearrangement of the enzyme structure. Hence, a single substitution in the binding site introduces a local conformational change that not only leads to very different catalytic properties, but can also significantly destabilize the protein.     

Extracellular acid proteases produced by Saccharomycopsis lipolytica.

Saccharomycopsis lipolytica CX161-1B produced at least three extracellular acid proteases during exponential growth in medium containing glycerol, Difco Proteose Peptone, and mineral salts at pH 3.4 (Difco Laboratories, Detroit, Mich.). Little extracellular acid protease activity was produced with glutamic acid as the sole nitrogen source, somewhat higher levels were obtained with peptone, and much higher levels were obtained with Difco Proteose Peptone. The relative amounts of the three proteases varied during growth on Difco Proteose Peptone, which suggested that the proteases were not coordinately regulated. The proteases were purified to near homogeneity (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) by use of ultrafiltration, gel filtration, and DEAE-Sephacel and hydroxylapatite chromatography. Protease I had a molecular weight near 28,000, an isoelectric point of pH 4.9, and a pH optimum of 3.5. Protease II had a molecular weight near 32,000 and a pH optimum of 4.2. Protease III had a molecular weight near 36,000, an isoelectric point of 3.8, and a pH optimum of 3.1. All three proteases were glycoproteins; proteases I, II, and III contained 25, 12, and 1.2% carbohydrate, respectively. The proteases were inhibited by pepstatin and 1,2-epoxy-3-(4-nitrophenoxy) propane and were largely insensitive to diazoacetyl-DL-norleucine methylester and to compounds which inhibit the serine, sulfhydryl, or metallo-proteases.     

Heterologous expression of heterodimeric laccase from Pleurotus ostreatus in Kluyveromyces lactis

Among the laccases produced by the white-rot fungus Pleurotus ostreatus, there are two closely related atypical isoenzymes, POXA3a and POXA3b. These isoenzymes are endowed with quaternary structure, consisting of two subunits very different in size. The POXA3 large subunit is clearly homologous to other known laccases, while the small subunit does not show significant homology with any protein in data banks. To investigate on the singular structure of the POXA3 complex, a new system for recombinant expression of heterodimer proteins in the yeast Kluyveromyces lactis has been set up. A unique expression vector has been used and the cDNAs encoding the two subunits have been cloned under the control of the same bi-directionally acting promoter. Expression of the large subunit alone and co-expression of both subunits in the same host have been demonstrated and the properties of the recombinant proteins have been compared. Clones expressing the large subunit alone exhibited always notably lower activity than those expressing both subunits. In addition to the activity increase, the presence of the small subunit led to a significant increase of laccase stability. Therefore, a role of the small subunit in POXA3 stabilisation is suggested.     

Enhancement of laccase production by Pleurotus ostreatus and its use for the decolorization of anthraquinone dye

The white-rot fungus Pleurotus ostreatus strain 32 is an excellent producer of the industrially important enzyme laccase. Laccase was the only ligninolytic activity detected in the supernatant when the fungus was grown in liquid culture with or without shaking. Growth and laccase production in static cultivation were superior to that in agitated cultivation, and N-limited culture is of benefit to laccase production. When using cellobiose and peptone as carbon and nitrogen source, a higher activity level was obtained. 2,2′-Azino-di-(3-ethylbenzothialozin-6-sulfonic acid) (ABTS) (1 mM) was shown to be the best inducer of laccase production, reaching maximum values of about 400 U/ml. Cu²⁺ (1 mM) also had a positive effect on laccase production, activity being enhanced to 360 U/ml. In addition, anthraquinone dye SN4R can be effectively decolorized by crude laccase (30 U/ml), the rate of which was 66%. The decolorization rate was increased by 90% with ABTS (0.16%) addition as a mediator of laccase.     

Production, purification and characterization of laccase from Pleurotus ostreatus grown on tomato pomace

A strain of Pleurotus ostreatus was grown in tomato pomace as sole carbon source for production of laccase. The culture of P. ostreatus revealed a peak of laccase activity (147 U/L of fermentation broth) on the 4th day of culture with a specific activity of 2.8 U/mg protein. Differential chromatographic behaviour of laccase was investigated on affinity chromatographic matrices containing either urea, acetamide, ethanolamine or IDA as affinity ligands. Laccase exhibited retention on such affinity matrices and it was purified on a Sepharose 6B—BDGE—urea column with final enzyme recoveries of about 60%, specific activity of 6.0 and 18.0 U/mg protein and purification factors in the range of 14–46. It was also possible to demonstrate that metal-free laccase did not adsorb to Sepharose 6B—BDGE—urea column which suggests that adsorption of native laccase on this affinity matrix was apparently due to the specific interaction of carbonyl groups available on the matrix with the active site Cu (II) ions of laccase. The kinetic parameters (V _max, K _m , K _cat, and K _cat/K _m ) of the purified enzyme for several substrates were determined as well as laccase stability and optimum pH and temperature of enzyme activity. This is the first report describing the production of laccase from P. ostreatus grown on tomato pomace and purification of this enzyme based on affinity matrix containing urea as affinity ligand.     

Classical breeding in Pleurotus ostreatus: a natural approach for laccase production improvement

White-rot basidiomycetes, the most common wood-rotting organisms, are characterized by their ability to produce extracellular oxidative enzymes, among which laccases are regarded as promising catalysts for many biotechnological applications. A significant obstacle to the exploitation of laccase-based bioprocesses is the large amounts of enzyme required. In this study the issue has been addressed by applying a classical breeding approach to increase laccase production yields in the white-rot fungus Pleurotus ostreatus. Starting from two different P. ostreatus varieties, three higher laccase-producing hybrids have been obtained by crossing selected compatible monokaryons. The three selected strains increased the titre of parental strains up to four fold, reaching an expression level of up to 100 000 U/L. One hybrid exhibited a more complex isoenzyme pattern, illustrating the potential of classical breeding to differentiate protein expression.     

Lovastatin production by Pleurotus ostreatus: effects of the C:N ratio

The types of carbon source and nitrogen source used as well as the C:N ratio in the medium influenced lovastatin production by Pleurotus ostreatus. The maximum value of the lovastatin yield was obtained in a medium that contained organic nitrogen.     

Cloning and sequencing of a laccase gene from the lignin-degrading basidiomycete Pleurotus ostreatus.

The gene (pox1) encoding a phenol oxidase from Pleurotus ostreatus, a lignin-degrading basidiomycete, was cloned and sequenced, and the corresponding pox1 cDNA was also synthesized and sequenced. The isolated gene consists of 2,592 bp, with the coding sequence being interrupted by 19 introns and flanked by an upstream region in which putative CAAT and TATA consensus sequences could be identified at positions -174 and -84, respectively. The isolation of a second cDNA (pox2 cDNA), showing 84% similarity, and of the corresponding truncated genomic clones demonstrated the existence of a multigene family coding for isoforms of laccase in P. ostreatus. PCR amplifications of specific regions on the DNA of isolated monokaryons proved that the two genes are not allelic forms. The POX1 amino acid sequence deduced was compared with those of other known laccases from different fungi.